Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis.
Recombinant beta-toxin has been expressed and secreted from Bacillus subtilis. Biological activity was tested in vivo and in vitro. The lethal dose in mice was determined. Hemolysis of rabbit and sheep erythrocytes was tested but no effect was observed. Seven mutant proteins were produced. Targets for mutagenesis were mostly selected on the basis of the similarity between beta-toxin and alpha-toxin from Staphylococcus aureus, a pore-forming toxin. Mutations of two amino acids affected the lethal dose in mice. Both residues have counterparts in the membrane binding region of alpha-toxin. Alteration of the single cysteine residue did not affect protein function, contrary to previous suggestions.
Steinthorsdottir
V
V
Institute for Experimental Pathology, University of Iceland, Reykjavik, Iceland. vstein@rhi.hi.is
Fridriksdottir
V
V
Gunnarsson
E
E
Andrésson
O S
OS
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics* / Q000494:pharmacology; D003016:Clostridium perfringens / Q000235:genetics*; D004252:DNA Mutational Analysis; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D006461:Hemolysis / Q000187:drug effects; D007928:Lethal Dose 50; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D012756:Sheep
D001427:Bacterial Toxins; C031884:CPB protein, Clostridium perfringens